The long term goal of this project is to develop vaccine molecular using a genetically detoxified bacterial exotoxin, pertussis toxin (PT*), as a vector molecule for induction of antigen-specific CD8+ T cell responses, including cytolytic T lymphocytes (CTL), which can protect against viral and parasitic infections. CTL recognize antigens as peptides in association with major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The candidate vaccine molecules will consist of genetically constructed fusions of peptide or polypeptide antigens with PT*, to stimulate antigen-specific CD8+ CTL responses in vivo. PT, like several other bacterial toxins, enters cells as part of its intoxication mechanism, and therefore can be exploited as an intracellular delivery molecule for antigens. We have found that PT* can deliver epitope peptides to MHC class I molecules in target cells for presentation to CTL, but that this apparently does not involve the endogenous cytosolic pathway of MHC class I antigen processing and presentation, to the endoplasmic reticulum (ER). We hypothesize that PT* can deliver epitomes to class I because of its retrograde transport in cells to the ER. We therefore aim to elucidate the intracellular transport pathway utilized by PT* to determine the mechanism by which it delivers epitopes to class I. This information will allow us to enhance this process, and therefore also to improve CTL responses, by modifying PT* to alter its intracellular transport. Such modified PT* molecules could represent powerful CTL-stimulating vaccine vector molecules. The fusion molecules will be tested in model systems in vitro and in vivo to assess their class I targeting capacity, their immunogenicity in vivo and their protective capacity in an animal model. The specific aims of this application are as follows. (1) To elucidate the intracellular transport pathway utilized by PT*. (2) To test and improve the capacity of PT*-epitope fusion molecules to stimulate epitope-specific CTL responses in vitro. (3) To test the capacity of PT*-epitope fusion molecules to stimulate epitope-specific CTL responses in mice and to protect mice from virus challenge. The advantages of PT* as a vaccine vector include its potent adjuvant-icity and its safety and non-reactogenicity for humans, being a component of a licensed pertussis vaccine.